Mix DNA into competent cells in a microcentrifuge.
Incubate the competent cell/DNA mixture on ice for 20-30 mins.
Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube
into a 42°C water bath for 30-60 secs
Put the tubes back on ice for 2 min.
Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and
grow in 37°C shaking incubator for 45 min.
Plate some or all of the transformation onto a LB agar plate containing
the appropriate antibiotic.
Incubate plates at 37°C overnight.
Verification of Transformation via Colony PCR
Put sample of bacteria colony into thermal cycler along with necessary
ingredients for PCR (dNTP, primers, buffer, Taq Polymerase) and heat to
95 C for 30 seconds.
Use the thermal cycler to achieve the following temperatures for 30 cycles
Use a sample of the PCR product to confirm length of the DNA insert via
gel electrophoresis
Exposure of E. Coli to PCB-11
Add 25 uL of PCB-11 to 250 uL of water and add this mixture to E. Coli plate.
Place E. Coli plate in 30℃ incubator for 24 hours.
Measurement of PCB Products in Wastewater Using
GCMS
Add 10 uL of PCB water mixture from E. Coli plate to a centrifuge tube and
spin down tube in centrifuge at 5,000 rpm for 10 min.
Add 100 uL of cyclohexane to GCMS tube and add 1 uL of water
from centrifuge tube.
Measure PCB concentration using GCMS (Gas Chromatography
Mass Spectrometry).
